Demonstration of chemotherapeutic mediated lymphatic changes in meningeal lymphatics in vitro, ex vivo, and in vivo.

Systemic chemotherapeutics target cancer cells but are also known to impact other cells away from the tumor. Questions remain whether systemic chemotherapy crosses the blood-brain barrier and causes inflammation in the periphery that impacts the central nervous system (CNS) downstream. The meningeal lymphatics are a critical component that drain cerebrospinal fluid from the CNS to the cervical lymph nodes for immunosurveillence. To develop new tools for understanding chemotherapy-mediated effects on the meningeal lymphatics, we present two novel models that examine cellular and tissue level changes. Our in vitro tissue engineered model of a meningeal lymphatic vessel lumen, using a simple tissue culture insert system with both lymphatic endothelial and meningeal cells, examines cell disruption. Our ex vivo model culturing mouse meningeal layers probes structural changes and remodeling, correlating to an explant tissue level. To gain a holistic understanding, we compare our in vitro and ex vivo models to in vivo studies for validation and a three-tier methodology for examining the chemotherapeutic response of the meningeal lymphatics. We have demonstrated that the meningeal lymphatics can be disrupted by systemic chemotherapy but show differential responses to platinum and taxane chemotherapies, emphasizing the need for further study of off-target impacts in the CNS.

Mitigating reactive oxygen species production and increasing gel porosity improves lymphocyte motility and fibroblast spreading in photocrosslinked gelatin-thiol hydrogels.

On-chip 3D culture systems that incorporate immune cells such as lymphocytes and stromal cells are needed to model immune organs in engineered systems such as organs-on-chip. Photocrosslinking is a useful tool for creating such immune-competent hydrogel cultures with spatial cell organization. However, loss of viability and motility in photocrosslinked gels can limit its utility, especially when working with fragile primary cells. We hypothesized that optimizing photoexposure-induced ROS production, hydrogel porosity or a combination of both factors was necessary to sustain cell viability and motility during culture in photocrosslinked gelatin-thiol (GelSH) hydrogels. Jurkat T cells, primary human CD4+ T cells and human lymphatic fibroblasts were selected as representative lymphoid immune cells to test this hypothesis. Direct exposure of these cells to 385 nm light and LAP photoinitiator dramatically increased ROS levels. Pretreatment with an antioxidant, ascorbic acid (AA), protected the cells from light + LAP-induced ROS and was non-toxic at optimized doses. Furthermore, scanning electron microscopy showed that native GelSH hydrogels had limited porosity, and that adding collagen to GelSH precursor before crosslinking markedly increased gel porosity. Next, we tested the impact of AA pretreatment and increasing gel porosity, alone or in combination, on cell viability and function in 3D GelSH hydrogel cultures. Increasing gel porosity, rather than AA pretreatment, was more critical for rescuing viability of Jurkat T cells and spreading of human lymphatic fibroblasts in GelSH-based gels, but both factors improved the motility of primary human CD4+ T cells. Increased porosity enabled formation of spatially organized co-cultures of primary human CD4+ T cells and human lymphatic fibroblasts in photo-crosslinked gels in a multi-lane microfluidic chip, towards modeling the lymphoid organ microenvironment. Some optimization is still needed to improve homogeneity between regions on the chip. These findings will enable researchers utilizing photocrosslinking methods to develop immunocompetent 3D culture models that support viability and function of sensitive lymphoid cells.

Modeling lymphangiogenesis: Pairing in vitro and in vivo metrics.

Lymphangiogenesis is the mechanism by which the lymphatic system develops and expands new vessels facilitating fluid drainage and immune cell trafficking. Models to study lymphangiogenesis are necessary for a better understanding of the underlying mechanisms and to identify or test new therapeutic agents that target lymphangiogenesis. Across the lymphatic literature, multiple models have been developed to study lymphangiogenesis in vitro and in vivo. In vitro, lymphangiogenesis can be modeled with varying complexity, from monolayers to hydrogels to explants, with common metrics for characterizing proliferation, migration, and sprouting of lymphatic endothelial cells (LECs) and vessels. In comparison, in vivo models of lymphangiogenesis often use genetically modified zebrafish and mice, with in situ mouse models in the ear, cornea, hind leg, and tail. In vivo metrics, such as activation of LECs, number of new lymphatic vessels, and sprouting, mirror those most used in vitro, with the addition of lymphatic vessel hyperplasia and drainage. The impacts of lymphangiogenesis vary by context of tissue and pathology. Therapeutic targeting of lymphangiogenesis can have paradoxical effects depending on the pathology including lymphedema, cancer, organ transplant, and inflammation. In this review, we describe and compare lymphangiogenic outcomes and metrics between in vitro and in vivo studies, specifically reviewing only those publications in which both testing formats are used. We find that in vitro studies correlate well with in vivo in wound healing and development, but not in the reproductive tract or the complex tumor microenvironment. Considerations for improving in vitro models are to increase complexity with perfusable microfluidic devices, co-cultures with tissue-specific support cells, the inclusion of fluid flow, and pairing in vitro models of differing complexities. We believe that these changes would strengthen the correlation between in vitro and in vivo outcomes, giving more insight into lymphangiogenesis in healthy and pathological states.

Engineering in vitro immune-competent tissue models for testing and evaluation of therapeutics.

Advances in 3D cell culture, microscale fluidic control, and cellular analysis have enabled the development of more physiologically-relevant engineered models of human organs with precise control of the cellular microenvironment. Engineered models have been used successfully to answer fundamental biological questions and to screen therapeutics, but these often neglect key elements of the immune system. There are immune elements in every tissue that contribute to healthy and diseased states. Including immune function will be essential for effective preclinical testing of therapeutics for inflammatory and immune-modulated diseases. In this review, we first discuss the key components to consider in designing engineered immune-competent models in terms of physical, chemical, and biological cues. Next, we review recent applications of models of immunity for screening therapeutics for cancer, preclinical evaluation of engineered T cells, modeling autoimmunity, and screening vaccine efficacy. Future work is needed to further recapitulate immune responses in engineered models for the most informative therapeutic screening and evaluation.

Modeling Immunity In Vitro: Slices, Chips, and Engineered Tissues.

Modeling immunity in vitro has the potential to be a powerful tool for investigating fundamental biological questions, informing therapeutics and vaccines, and providing new insight into disease progression. There are two major elements to immunity that are necessary to model: primary immune tissues and peripheral tissues with immune components. Here, we systematically review progress made along three strategies to modeling immunity: ex vivo cultures, which preserve native tissue structure; microfluidic devices, which constitute a versatile approach to providing physiologically relevant fluid flow and environmental control; and engineered tissues, which provide precise control of the 3D microenvironment and biophysical cues. While many models focus on disease modeling, more primary immune tissue models are necessary to advance the field. Moving forward, we anticipate that the expansion of patient-specific models may inform why immunity varies from patient to patient and allow for the rapid comprehension and treatment of emerging diseases, such as coronavirus disease 2019.

Endothelial cell crosstalk improves browning but hinders white adipocyte maturation in 3D engineered adipose tissue.

Central to the development of adipose tissue (AT) engineered models is the supporting vasculature. It is a key part of AT function and long-term maintenance, but the crosstalk between adipocytes and endothelial cells is not well understood. Here, we directly co-culture the two cell types at varying ratios in a 3D Type I collagen gel. Constructs were evaluated for adipocyte maturation and function and vascular network organization. Further, these constructs were treated with forskolin, a beta-adrenergic agonist, to stimulate lipolysis and browning. Adipocytes in co-cultures were found to be less mature than an adipocyte-only control, shown by smaller lipid droplets and downregulation of key adipocyte-related genes. The most extensive vascular network formation was found in the 1:1 co-culture, supported by vascular endothelial growth factor (VEGF) upregulation. After forskolin treatment, the presence of endothelial cells was shown to upregulate PPAR coactivator 1 alpha (PGC-1α) and leptin, but not uncoupling protein 1 (UCP1), suggesting a specific crosstalk that enhances early stages of browning.